Protocol 35033: Probe Assay - Hesx1<em1(IMPC)J>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

>chr14:27001516+27001627 112bp GAGAATTGAGTTGGTACCGAGGA GTCTGGCTTCTGTCACAACCT

Mut= 120 bp

Wt= 112 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TTCTGTTCTGTATCTATAAAAGGTCTCCAACAGGGTCGGAAGGGAATAGAGTGCCTGCCCTAGGTGGTTCACTCCCAGTGAGAAATAGTATTTGTTTCAGAAGACCCTCTGTGTACATGTGTCACGTCAGGCATTCTAAATCTCTCCTcactgaaaaatgaatggtagagttcaaaatgtatgttctctatcaccaaggcttctctctctctctctctctctctctccctctctctcccttcccccatcctctttttttttgggggggggggaatgggggagacaattcttttgtgaaaccttgaagaattccctaggaaaattcaaagcaagttactgaaatgacttttttccagaactttgtggaacataggcaatataagatggaccttcttcttctgattttagagaaaggtggcaacccacccctacatgccccagatcttcccagtgagacttcatttccttgtccagtggatcatccaaggccagaagaaagggctccgaaatatgaaaattatttttcagcctccgaaacacgctctttgaaaagagaattgagttggtaccgaggacgaaggccaagaaccgctttcacccagaaccaggtgagaagatttttcagccaatagtgacaaCACAAAGGTTGTGACAGAAGCCAGACACTCTGGGGTCCCAGTGTAGAAGTGGCATTAGTGAAAGGGTGGACTCATAGTTTAACAGTCTCTGGGCAACCAATTGTTTTAAAATGCTTTTGGATTAAAGGCCCAAATTTAAGTGCATTTTTCATTTGAAAGCCCAATT

This mutation is a 490 bp deletion beginning at Chromosome 14 position 27,001,112 bp and ending after 27,001,601 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45083 GAG AAT TGA GTT GGT ACC GAG GA Wild type Forward A
45084 GTC TGG CTT CTG TCA CAA CCT Common A
45085 CTG CCC TAG GTG GTT CAC TC Mutant Forward A
45086 Fluorophore-1 AGG CCA AGA ACC GCT TTC Quencher-1 WT Probe
45087 Fluorophore-2 CAT GTG TCA CGT CAG GCA TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45083 0.40 uM
45084 0.40 uM
45085 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.